This SBIR project seeks to develop novel technologies for the manipulation and labeling of the interior of living eukaryotic cells for research, diagnostic and eventually therapeutic purposes. Cell-penetrating peptides offer the tantalizing prospect of delivering a broad palette of user-defined protein cargoes through the plasma membranes of eukaryotic cells. Cargo protein delivery via CPPs has many advantages over transfection with respect to variety of deliverable biomolecules, rapidity of protein expression/delivery and control of protein levels over time. Current CPP technologies are either cumbersome and require the production of individually labeled proteins, or suffer from a lack of specificity and low efficiency. Our innovative core technology is based on cell-penetrating peptide-coupled adaptor proteins that allow the spontaneous loading of almost any desired cargo protein for rapid delivery into cells. Our CPP-adaptor technology not only allows delivery of an extended array of cargoes, but has advantages in speed and ease of use as well as safety enhancements compared to current technologies. In addition, cargoes are released from the adaptors in the cytoplasm and the system and can be produced as a modular kit allowing affinity cargo protein purification using the same tag recognized by the CPP-adaptor. Additional features we seek to develop will allow subcellular delivery based on the properties of different CPPs (e.g. nuclear, mitochondrial or lysosomal localization) and enable simultaneous delivery of multiple cargoes to different compartments.